Small, volatile and non-volatile organic molecules, commonly referred to as pheromones, mediate species-specific chemical communication between terrestrial animals. Pheromones are present in the secretions and excretions from various organs and tissues, including the skin, and represent diverse families of chemical structures. Pheromones play essential roles in sexual activity, reproductive biology, and other innate animal behaviors (Luscher et al., (1959) Nature 18:55–56; Meredith (1983) in Pheromones and Reproduction in Mammals (Vandenbergh, ed.) pp. 199–252, Academic Press; Stern et al., (1998) Nature 392:177–179; Wysocki, (1979) Neurosci. Biobehav. Rev. 3:301–341; Jacob et al., (2000) Hormones and Behavior 37:57–78; Grosser et al., (2000) Psychoneuroendocrinology 25:289–299). Some, but not all, terrestrial vertebrates detect pheromones in the vomeronasal organ (the VNO), also known as Jacobson's organ, a small dead-end tubular structure with an opening into the nasal cavity that is located bilaterally at the base of the nasal septum (Moran et al., (1991) J. Steroid Biochem. Molec. Biol. 39:545–552.).
The VNO was first identified in humans in 1703 but it was believed to be a vestigial organ without function in the adult. In the 1990s, the presence of a VNO was established, caudal to the nasal septal cartilage on both sides of the nasal septum, in more than 1700 normal male and female human subjects (Berliner, (1996) J. Steroid Biochem. Molec. Biol. 58:1–2; Gaafar et al., (1998) Acta Otolargyngol. 118:408–412; Smith et al., (1998) Micro. Res. Tech. 41:483–491) The VNO is physically separate and functionally distinct from the olfactory epithelium that detects the volatile odorants. Odorants do not bind to the VNO receptors.
The VNO is lined with neuroepithelial cells with a microvillar surface that is the presumptive site of pheromone receptors. Immunohistochemical staining of adult human VNO epithelium detects neuron-specific enolase and protein gene product (PGP) 9.5, both neuronal and neuroendocrine markers, in some bipolar cells with morphological similarities to olfactory receptor neurons (Takami et al., (1993) Neuroreport 4:375–378). More recent studies show that the majority of the cells lining the lumen of the human VNO stain with antibodies to synaptophysin or chromogranin which are also markers for neuronal and neuroendocrine cells. These data provide clear evidence for the existence of a neuroepithelium in the human VNO. However, Takami et al. (1993) do not detect olfactory marker protein (OMP) in the human VNO even though it is expressed in the VNO of other vertebrates including rodents. This may reflect an important and interesting species difference between humans and other vertebrates.
In animals, signals from the olfactory epithelium travel via the olfactory bulb to the olfactory cortex and then on to other regions of the brain. In contrast, signals from the VNO are transmitted through the accessory olfactory bulb to the amygdala and hypothalamus (Broadwell et al., (1975) J. Comp. Neurol. 163:329–346; Kevetter et al., (1981) J. Comp. Neurol. 197:81–98). Surgical ablation of the VNO in male rodents alters a variety of endocrine-mediated responses to female pheromones including androgen surges, vocalization, territorial marking, and inter-male aggression. Ablation of the VNO in female rodents delays or prevents activation of reproduction, abolishes the effects of over-crowding on. sexual maturation, and reduces maternal responses to intruders (Wysocki et al., (1991) J. Steroid Biochem. Molec. Biol. 39:661–669). In humans, the defect(s) that causes the inherited hypogonadal disorder, Kallmann Syndrome, is also associated with defective development of the VNO-terminalis complex (Kallmann et al., (1943) Am. J. Ment. Defic. 48:203–236).
Application of only femtomole quantities of any of several proprietary, synthetic vomeropherins directly to the VNO of human volunteers rapidly induces reproducible negative voltage potentials that can be measured locally with a multifunctional miniprobe. The electrophysiological response in the VNO is characteristic of a mass receptor potential. The magnitude of the response is dose-dependent and is accompanied by changes in autonomic nervous system function, brain wave activity, gonadotropin secretion, and mood (Berliner et al., (1996) J Steroid Biochem, Molec. Biol. 58:259–265; Monti-Bloch et al. (1998a) J. Steroid Biochem. Molec. Biol. 65:237–242; Monti-Bloch et al., (1998b) Ann. N.Y. Acad. Sci. 855:373–389; Monti-Bloch et al., (1994) Pyschoneuroendocrinology 19:673–686; Monti-Bloch et al., (1991) J. Steroid Biochem. Molec. Biol. 39:573–582; Grosser et al., (2000) Psychoneuroendocrinology 25:289–299).
Recent FMRI studies detect dose-dependent activation of the anterior medial thalamus, the inferior frontal gyrus, and other regions of the human brain, in the absence of detectable odor, following administration of estra-1,3,5(10),16-tetraen-3yl acetate (PH15) to human volunteers. Although Sobel et al. ((1999) Brain 122:209–217) deliver the compound non-specifically to the nasal cavity in these fMRI tests, Monti-Bloch et al. (1994) have demonstrated that this compound induces physiological responses in vivo only when applied specifically to the VNO but not when applied to either olfactory or respiratory epithelium of human subjects. Therefore, the fMRI data support the existence of a functional neurological connection between the VNO and the human brain which can be activated by a vomeropherin.
Administration of naturally occurring compounds of known structure such as estra-1,3,5(10),16-tetraen-3-ol and androsta-4,16-dien-3-one to the human VNO induce bradycardia, bradypnea, increases in core body temperature, and other physiological responses. Stern et al. (1998) have demonstrated that odorless human pheromones, obtained from the axillae of women at different stages of the menstrual cycle, exert opposing effects on ovulation when applied above the lips where they can volatilize into the nasal cavity of recipient females. Some vomeropherins act exclusively in human females or in males, and others exert opposite effects on autonomic reflexes such as body temperature. Taken together, these data provide substantial support for the existence of a functional VNO in humans with the capacity to exert significant physiological effects in vivo.
The VNO system affords the unique opportunity to develop and market novel therapeutics to treat disease via previously unexploited targets and neurological pathways. This approach has substantial benefits for the patient over existing therapies including: (i) the ease of delivery to the VNO, (ii) the requirement for only picograms of drug, (iii) the rapid response to drug, and (iv) the apparent absence of the side-effects and toxicity frequently associated with systemic (e.g., oral) delivery of drug. Thus, targeting receptors in the human VNO for the treatment of disease is desirable.
The standard bioassay for screening candidate vomeropherins requires the participation of human volunteers because pheromones are species-specific. In this assay, the compounds are delivered directly to the VNO of volunteers under IRB-approved protocols, thus necessitating prior toxicological study of each candidate vomeropherin in rodents. This expensive and time-consuming process limits the number of compounds that can be tested and hampers the detailed structure-activity relationship (SAR) analyses that are essential to successful drug discovery.
Viable neuroepithelial cells may be harvested directly from the human VNO for testing in vitro. The harvested VNO cells retain their characteristic neuroepithelial morphology in culture and respond electrophysiologically to the application of vomeropherins in vitro, thereby demonstrating the existence of functional receptors in cells from the target tissue. Although this method still requires the participation of human volunteers, it increases the screening throughput and decreases the number of animals required for toxicological studies. However, only a limited number of non-dividing cells with a ˜2-week life-span are obtained from each volunteer, and thus we require an entirely new approach to meet the demands of modem high throughput drug screening and SAR.
Several groups have cloned receptor cDNAs that are expressed exclusively in the VNO of rats and mice, but, to date, no one has cloned human VNO receptor cDNAs. The sequence of the cloned rodent receptor cDNAs indicates that they belong to the superfamily of G protein-coupled receptors containing seven transmembrane domains, but they are unrelated to any of the G protein-coupled receptors expressed in the olfactory epithelium (Dulac et al, (1995) Cell 83:495–206; Herrada et al., (1997) Cell 90:763–773; Matsunami et al., (1997) Cell 90:775–784; Ryba et al., (1997) Neuron 19:371–379; Saito et al., (1998) Brain Res. Molec. Brain Res. 60:215–227). Database comparisons identify motifs common to Ca2+-sensing and metabotropic glutamate receptors in some of the clones. The apparent lack of homology to olfactory receptors is consistent with the observation that many vomeropherins are inactive when applied specifically to human olfactory epithelium in vivo.
Each cloned rodent receptor messenger RNA (mRNA) is detected by in situ hybridization in only a small number of neuroepithelial cells that are dispersed throughout the rodent VNO, and it is likely that each cell expresses only a single receptor gene. (Dulac et al., 1995; Herrada et al., 1997; Matsunami et al., 1997; Ryba et al., 1997; Saito et al., 1998). Some of the cloned rodent receptors exhibit sexually dimorphic expression, i.e., they are expressed differently in males or females.
The rodent VNO receptors are assigned to separate multi-gene families by two criteria: (i) the length of the extracellular (N-terminal) protein domain, and (ii) the isoform of the signal-transducing G protein co-expressed in the same cell. Receptors in the “V1R” family have a relatively short extracellular N-terminal domain and are expressed primarily in cells that express a Gαi isoform of G protein. Receptors in the “V2R” family have a long extracellular N-terminal domain and are expressed primarily in cells that express a Gα0 isoform of G protein. Differences at the N-terminus between the V1R and V2R families may reflect differences in the structure of the ligand and/or in the location of the ligand-binding domain. (Matsunami et al., 1997; Ryba et al., 1997; Krieger et al., (1999 J. Biol. Chem. 274:4656–4662). Neuroepithelial cells expressing these distinct G protein isoforms are spatially segregated in the VNO in separate apical and basal longitudinal zones, suggesting that there is true physiological significance to the differences between the V1R and V2R receptor families.
Krieger et al. (1999) have recently shown that G protein-coupled receptors expressed in the rodent VNO are functionally linked to signal transduction pathways. Their results demonstrate that volatile and non-volatile pheromonal components of male rat urine selectively activate the major Gα protein subtypes (Gi and G0, respectively) expressed in the VNO of female rats. The data imply that V1R family receptors, which are co-expressed with Gi, respond to volatile compounds whereas V2R family receptors, which are co-expressed with G0, respond to non-volatile protein components of urine.
Dulac and Axel (1995) estimate that, in total, the rat V1R family contains approximately 35 candidate pheromone receptors; Herrada and Dulac (1997) and Ryba and Tirindelli (1997) estimate that the rat V2R family contains an additional 100 receptors. Of the various rodent tissues tested, only mRNA from the VNO gives a positive signal on northern blots probed with the cloned (32P-labeled) pheromone receptor cDNAs. At this limit of sensitivity, these results suggest that the pheromone receptors are expressed exclusively (primarily) in the VNO. At the present time, it is not known if each VNO receptor recognizes a distinct pheromone or if several receptors recognize the same compound.
At reduced stringency, the cloned rodent VNO receptor cDNAs cross-hybridize to human genomic DNA. Dulac and Axel (1995) detect approximately 15 human genes that cross-hybridize to rat V1R family probes, and Herrada and Dulac (1997) detect an additional ten human homologues that cross-hybridize to rat V2R family probes. The two sequenced human V1R genomic DNA clones have ˜40–50% identity with the closest rat homologue. However, both human genomic clones have a stop codon in the putative coding region and may thus be pseudogenes (Dulac and Axel, 1995). Nevertheless, cross-hybridization suggests the evolutionary conservation of G protein-coupled receptors in the VNO and thereby provides a means to isolate human receptor clones.
The presence of these pseudogenes does not preclude the existence of functional human VNb receptor genes, especially in view of our assays with cells harvested directly from the VNO (Monti-Bloch (1997) Chemical Senses 22:752). The past difficulties in isolating, characterizing and cloning a VNO receptor reinforce our assertion that an appropriate way to isolate functional clones of the human VNO receptors is via cDNA prepared directly from the target tissue. In fact, Cao et al. ((1998) Proc. Nad. Acad. Sci. USA 95:11987–11992) have successfully isolated homologues from a goldfish cDNA library using probes based on the rodent receptor sequences even though that species lacks a defined VNO. The presence of pseudogenes in the family has not prevented the successful cloning of olfactory or VNO receptors from a variety of species and they should present no greater obstacle to the cloning of human VNO receptors.
Thus, isolation and characterization of the human VNO receptors is desirable for the development of new drugs, high throughput assays and characterization of the receptors and their signal transduction pathways.